We are interested in the understanding the specificity of protein-ligand interactions and the microevolutionary processes that permit and promote changes at the protein-ligand interface. To explore these issues, we are studying the Phd repressor/antitoxin and selected homologs. Phd is a small 73 amino acid protein with a remarkable number of ligands. It dimerizes, it binds to operator DNA (and thus represses transcription), and it binds (and thus neutralizes) the toxin. In the presence of toxin, dimers of Phd bound to adjacent DNA sites engage in cooperative (tetrameric) interactions, involving yet another macromolecular contract. Lastly, Phd binds to (and is degraded by) the CIpXP protease. A homologous protein from Salmonella, engages in a similar set of interactions, but with somewhat different specificities. We have identified five amino acid residues that are specifically involved in repression and another four amino acids that are specifically involved in the neutralization of toxin. Here, we propose to extend our analysis to identify additional participants in the repressor-operator, toxin-antitoxin, and oligomeric interactions. The specific aims are 1) Amino acid residues in Phd and Doc that contribute to toxin-antitoxin recognition. 2) Amino acid residues and nucleotides that contribute to repressor-operator recognition. 3) Amino acid residues in Phd that contribute to the (Doc-mediated) tetramerization of Phd. We will use bioinformatics tools, site-directed mutagenesis (alanine scans, charge reversals and phylogenic substitutions) and classical genetics to identify point mutations that disrupt a protein-ligand interaction or alter the specificity of that interaction. By testing each mutant for multiple activities, we will be able to determine whether the effects of the mutation are local (affecting a single activity of Phd) or global (affecting multiple activities of Phd). The long-term objective of this research is to understand protein-ligand interactions well enough to recognize ligand binding domains, match proteins to their preferred ligands, design proteins to bind specific ligands and design ligands to bind specific proteins. A superior understanding protein-ligand interactions will assist in the rational design of drugs (ligands) that interact with specific protein targets (agents of disease).